Treatment of skin with adenosine or adenosine analog

ABSTRACT

Methods for enhancing the condition of non-diseased skin by application of compositions containing adenosine or an adenosine analog are disclosed. Also disclosed are methods for increasing DNA synthesis or protein synthesis in dermal cells, and methods for increasing dermal cell size, by application of compositions containing adenosine.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Work on this invention was supported by funds from the United Statesgovernment (Public Health Service Grants HL-22828 and AG-11491). Thegovernment therefore has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to dermatology and cell biology.

BACKGROUND OF THE INVENTION

Skin includes a surface layer, known as the epidermis, and a deeperconnective tissue layer, known as the dermis. The epidermis undergoescontinuous turnover as the outermost cells are exfoliated and replacedby cells that arise from inner dermal layers. The dermis is composed ofa variety of cell types, including fibroblasts.

Skin thickness begins to decline in humans after the age of 20 as thedermis becomes thinner and the number of skin fibroblasts declines. Asskin ages, or is exposed to UV light and other environmental insults,changes in the underlying dermis can lead to the functional andmorphological changes associated with damaged skin. Decreases in theabundance and function of products of the fibroblasts, which includecollagen and proteoglycans, are believed to play major roles in wrinkledand damaged skin.

SUMMARY OF THE INVENTION

We have discovered that adenosine stimulates DNA synthesis, increasesprotein synthesis, and increases cell size in cultures of human skinfibroblasts. Based on this discovery, the invention provides methods andcompositions for enhancing the condition of skin.

In general, the invention provides a method for enhancing the conditionof non-diseased skin of a mammal, e.g., a human. The method includestopically applying a therapeutically effective amount of a compositionincluding adenosine or an adenosine analog to non-diseased skin of themammal.

The invention also provides a method for promoting healing of broken,non-diseased skin in a mammal by is topically administering acomposition including a therapeutically effective amount of adenosine oran adenosine analog to the mammal.

Also included in the invention is a method for increasing DNA synthesisin a dermal cell of non-diseased skin of a mammal. The method includestopically administering a therapeutically effective amount of adenosineor an adenosine analog to a region of non-diseased skin of the mammalcontaining dermal cell. The adenosine is added so that it does not causeproliferation of the dermal cell.

The invention also features a method of increasing protein synthesis ina dermal cell of non-diseased skin of a mammal. The method includestopically administering a composition including a therapeuticallyeffective amount of adenosine or an adenosine analog to a region of skinof the mammal containing the dermal cell. The adenosine or adenosineanalog does not cause proliferation of the dermal cell.

Also provided in the invention is a method of increasing cell size in adermal cell in non-diseased skin of a mammal, e.g., a human. The methodincludes topically administering a composition including atherapeutically effective amount of adenosine to a region of skin of themammal containing the dermal cell, wherein addition of the adenosinedoes not cause proliferation of the dermal cell, wherein addition of theadenosine does not cause proliferation of the dermal cell.

The invention also includes a method for enhancing skin condition in amammal, e.g., a human. The method includes providing fibroblasts fromthe mammal ex vivo, culturing the fibroblasts in the presence ofadenosine, and reintroducing the fibroblasts into the mammal.

The therapeutically effective amount of adenosine used in theabove-described methods is preferably 10⁻³ M to 10⁻⁷ M, more preferably10⁻⁴ M to 10⁻⁶ M, and most preferably about 10⁻⁴ M.

The composition used in the above-described methods can include a secondagent in addition to adenosine. The second agent can be, e.g. an agentthat promotes binding of adenosine or an adenosine analog to anadenosine receptor, an angiogenic factor such as vascular endothelialcell growth factor (VEGF), basic fibroblast growth factor (BFGF), anagent that itself enhances skin condition, such as tretoinin or anotherknown conditioning agent such as an emollient, a humectant, or anocclusive agent.

In preferred embodiments of the invention, the adenosine or an adenosineanalog does not promote skin cell proliferation.

The invention also provides a composition including about 10⁻³ M toabout 10⁻⁷ M adenosine and a therapeutically effective amount of anangiogenesis factor. In some embodiments, the composition of theadenosine is about 10⁻⁴ M.

As used herein, “enhancement of skin condition” means a noticeabledecrease in the amount of wrinkling, roughness, dryness, laxity,sallowness, or pigmentary mottling in skin.

As used herein, a “therapeutically effective amount” of adenosine or anadenosine analog means an amount that enhances skin condition whenapplied to skin.

As used herein, “non-diseased skin” means skin free of any proliferativedisorder observable by visual inspection.

The present invention advantageously allows for enhancement of skincondition. This results in skin that shows a less wrinkled, rough, ordry complexion. For example, the invention provides for enhancing thecondition of skin damaged due to exposure to the sun or skin whosecondition has deteriorated due to normal aging.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of this invention will be apparent fromthe following description of the preferred embodiments thereof, and fromthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are histograms showing the effect of adenosine on[³H]thymidine incorporation in cultures of normal human skin (FIG. 1A)and lung fibroblasts (FIG. 1B). After incubation in serum-free mediumfor 24 hours, cells were exposed to 10⁻⁴ M adenosine for 18 hours.Medium was replaced with serum-free medium without adenosine, and[³H]thymidine was added. Results are expressed as percent [³H]thymidineincorporation compared to control cultures without adenosine and aremeans±SEM for 4-5 experiments. “*” denotes value was significantlydifferent from control value without adenosine.

FIGS. 2A and 2B are histograms showing concentration responses ofadenosine-stimulated protein synthesis in human skin fibroblasts from ayoung (FIG. 2A) and aged (FIG. 2B) donor. Cells were grown to 75%confluence. Medium was then replaced with serum-free medium with orwithout adenosine. After 48 hours, [³H]phenylalanine incorporation wasdetermined as described. Results are expressed as % [³H]phenylalanineincorporation compared to control cultures without adenosine and aremeans±SEM for 6-25 experiments. “*” denotes value was significantlydifferent from control value without adenosine.

DETAILED DESCRIPTION

The invention is suitable for treating skin of a mammal, e.g., a human,for which promotion of fibroblast-associated dermal functions isdesired. For example, promotion of fibroblast-associated functions isdesirable in enhancing the condition of aged skin, which is associatedwith a decrease in dermal cell function and is characterized byincreased dryness or roughness, or both. The method can also be used onsubjects having otherwise damaged skin, e.g., wrinkled skin and skinwith a non-proliferative disorder. The method can may further be usedprophylactically on a subject to minimize deterioration of skincondition associated with aging or environmental factors, such asphotodamage.

Adenosine and suitable adenosine analogs are suitable for use inenhancing skin condition. Adenosine analogs such as adenosine agonists,adenosine receptor agonists, and compounds that increase intracellularor extracellular adenosine levels are suitable for use in the invention.

Agonists of adenosine include 2′-deoxyadenosine; 2′,3′-isopropoylideneadenosine; toyocamycin; 1-methyladenosine; N-6-methyladenosine;adenosine N-oxide; 6-methylmercaptopurine riboside; 6-chloropurineriboside, 5′-adenosine monophosphate, 5′-adenosine diphosphate, or5′-adenosine triphosphate. Adenosine receptor agonists includephenylisopropyl-adenosine (“PIA”), 1-Methylisoguanosine, ENBA (S(−),N⁶-Cyclohexyladenosine (CHA), N⁶-Cyclopentyladenosine (CPA),2-Chloro-N₆-cyclopentyladenosine, 2-chloroadenosine, and adenosine aminecongener (ADAC), all of which are agonists for the adenosine A₁receptor. Other receptor agonists include 2-p-(2-carboxy-ethyl)phenethyl-amino-5′-N-ethylcarboxamido-adenosine (CGS-21680),N-ethylcarboxamido-adenosine (NECA) and napthyl-substitutedaralkoxyadenosine (SHA-082), 5′ (N-cyclopropyl)-carboxamidoadenosine,DPMA (PD 129,944), Metrifudil, which are agonists for the adenosine A₂receptor. Other adenosine receptor agonists include those whichpreferentially bind the A₁ receptor relative to the A₂ receptor, such as2-Chloroadenosine, N⁶-Phenyladenosine, and N⁶-Phenylethyladenosine; andthose which preferentially bind the A₂ receptor relative to the A₁receptor, such as 2-Phenylaminoadenosine and MECA.

Also suitable for use are compounds that increase intracellularadenosine concentration by inhibiting the cellular uptake of adenosineor the breakdown of adenosine. One pathway of adenosine metabolism isthe conversion of adenosine to inosine by adenosine deaminase. Anexample of an adenosine deaminase inhibitor iserythro-9-(2-hydroxy-3-nonyl) adenine (“EHNA”). Adenosine kinaseinhibitors can also be used. Adenosine kinase converts adenosine toadenosine monophosphate by adenosine kinase. An example of an adenosinekinase inhibitor is iodotubercidin. Other suitable compounds includethose that inhibit the dipyridamole-sensitive nucleoside transporter,which exports adenosine from the cytoplasm, and agents that promote theactivity of a 5′-nucleotidase, e.g., the ATP-activated 5′-nucleotidase,which forms adenosine. Compounds that increase tissue adenosine and ATPlevels include acadesine (AICA-riboside), which is described in Gruberet al., Circulation 80:1400-1411 (1989).

Adenosine can be also be administered with a second compound. The secondcompound can enhance the action of adenosine or the adenosine analog,e.g., by enhancing binding of adenosine or an adenosine analog to anadenosine receptor. An example of such a compound is PD 81,728, which isdescribed in Kollias-Baker et al. J. Pharmacol. Exp. Ther. 281:761-68.Alternatively, the second agent can itself act to enhance skincondition. Examples of these types of agents include tretinoin, arecognized skin conditioning agent (see, e.g., Olsen et al., J. Amer.Acad. Dermatol. 37:217-26, 1997), an angiogenic factor such as vascularendothelial cell growth factor (VEGF) or basic fibroblast growth factor(BFGF), or a conditioning agent.

The second compound can also be a conditioning agent such as anemollient, humectant, or occlusive agent. Numerous examples ofparticular conditioning agents are provided in the CTFA CosmeticIngredient Handbook (Cosmetic Toiletries and Fragrances Association,Washington, D.D., 1988). Emollients help to maintain the soft, smooth,and pliable appearance of skin and function by remaining on the skinsurface or in the stratum corneum to act as lubricants, to reduceflaking, and to improve the skin's appearance. Examples of emollientsinclude acetyl trioctyl citrate, cetyl alcohol, butyl myristate, cetylalcohol, and mineral oil.

Humectants act to increase the water content of the top layers of theskin. Humectants include, e.g., acetamide MEA, fructose, and xylitol.Occlusive agents inhibit the evaporation of water from skin, therebyincreasing the water contend of the skin. Acetylated castor oil, mineraloil, and lauryl stearate are examples of occlusive agents.

A subject can be treated by applying adenosine or an adenosine analog ina pharmaceutical composition in an effective amount and for a period oftime sufficient to improve the condition of the skin.

The pharmaceutical composition may be formulated using conventionalmethods to prepare pharmaceutically useful compositions. Suchcompositions preferably include at least one pharmaceutically acceptablecarrier, such as those described in Remington's Pharmaceutical Sciences(E. W. Martin). In addition, the compositions preferably include apharmaceutically acceptable buffer, preferably phosphate bufferedsaline, together with a pharmaceutically acceptable compound foradjusting isotonic pressure, such as, for example, sodium chloride,mannitol, or sorbitol.

Adenosine or an adenosine agonist can also be provided in carriers andadjuvants such as ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances, such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicatei polyvinyl pyrrolidone, cellulose-basedsubstances and polyethylene glycol. Adjuvants for topical or gel baseforms of adenosine or adenosine analogs may, for example, be selectedfrom the group consisting of sodium carboxymethylcellulose,polyacrylates, polyoxythylene-polyoxypropylene-block polymers,polyethylene glycol and wood wax alcohols. For all administrations,conventional depot forms may be used.

The adenosine or adenosine analog-containing compositions may be in anypharmaceutically acceptable dosage form. They are preferably applied bytopical routes to exert local therapeutic effects. For topicalapplication, the penetration of the adenosine into skin tissue may beenhanced by a variety of methods known to those of ordinary skill in theart. For example, adenosine may be applied directly and mechanicallyrubbed into the skin. Alternatively, adenosine or adenosine analogs maybe incorporated into a transdermal patch that is applied to the skin.Preferably, the penetration resulting from these methods is enhancedwith a chemical transdermal delivery agent such as dimethyl sulfoxide(DMSO) or the nonionic surfactant, n-decylmethyl sulfoxide (NDMS), asdescribed in Choi et al. Pharmaceutical Res., 7(11):1099, 1990.

Other modes of administration include, e.g., oral, subdermal,intradermal, or intravenous. When oral administration is used, it iscritical that the adenosine or adenosine analog be delivered to that itis not degraded prior to exiting the digestive system.

The most effective mode of administration and dosage regimen ofadenosine or the adenosine analog will depend upon the skin condition,previous therapy, the subject's health status, response to theadenosine, the judgment of the treating physician and the mode in whichthe adenosine is applied. For example, dosages for a therapeuticallyeffective amount for topical application would be in the range of 100 ngto 10 mg per treated surface area per day. The adenosine may beadministered to the patient at one time or over a series of treatments.When adenosine or the adenosine analog is administered in conjunctionwith a second agent, they can be administered either concurrently orsequentially, and can be administered in the same mode or a differentmode, e.g., topical or oral.

Adenosine or an adenosine analog enhances skin condition when there is anoticeable decrease in noticeable decrease in the amount of wrinkling,roughness, dryness, laxity, sallowness, or pigmentary mottling of thetreated skin. Methods of measuring improvements in skin condition arewell known in the art (see, e.g., Olsen et al., J. Amer. Acad. Dermatol.26:215-24, 1992), and can include subjective evaluations by the patientor a second party, e.g., a treating physician. Objective methods caninclude skin topography measurements, such as those described in Groveet al., J. Amer. Acad. Dermatol. 21:631-37 (1989). In skin topographymeasurements, silicone rubber replicas are made of a small area of skin,e.g., a 1 cm diameter circular area. The silicone rubber replicascapture fine lines and wrinkles on the skin. These specimens are thenanalyzed using computerized digital image processing to provide anobjective measurement of the skin's topography. Skin topographymeasurements generated following digital-image processing can bemeasured using the values R_(a) and R_(z) as described in Olsen et al.,J. Amer. Acad. Dermatol. 37:217-26, 1997, where R_(a) represents thearea of deviation of skin surface features above and below an averagecentral line, and R_(z) represents the difference between the maximumand minimum heights in five equal segments of the skin surface profile.A statistically significant decline (e.g., P<0.05) in R_(a) and R_(z)values in skin treated with adenosine or an adenosine analog compared tountreated skin indicates an enhancement of skin condition.

Fibroblasts treated with adenosine or adenosine analogs can also beincorporated into a matrix and implanted in the body, e.g., as part of askin graft. In addition, fibroblasts can be genetically engineered exvivo to increase the amount of intracellular adenosine levels and thenre-introduced into a human patient. (See, for example, Anderson et al.U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959,each of which is incorporated by reference herein in its entirety).

Experimental Information

Cell Culture

Human skin fibroblasts and human lung fibroblasts were supplied by theN.I.A. Aging Culture Repository Center (Camden, N.J.). For skinfibroblasts, primary cultures had been initiated from explants obtainedfrom a 3 mm punch biopsy of the mesial aspect of the upper left arm.Human lung fibroblasts (IMR-90) were established from a 16-week normalfemale fetus. All cells displayed a normal diploid karyotype and allcells tested negative for bacteria, fungi and mycoplasma contamination.

Cells were grown in Eagle's minimal essential medium (MEM) supplementedwith 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/mlstreptomycin in a 37° C., 5% CO₂/95% air environment. After reachingconfluence, cells were subcultivated with 0.25% trypsin in MEM with noadded Ca²+ or Mg²⁺.

Incorporation of [³H]Thymidine

As an index of DNA synthesis incorporation of [³H]thymidine was measuredas described in Ethier et al., Am. J. Physiol. 272:H1470-79 (1997).Confluent monolayers of human skin fibroblasts in MEM plus 10% FBS wereseeded into 16 mm diameter culture wells (24-well plates) at a densityof 1×10⁴ cells/cm². Cells were grown at 37° C. under standard cultureconditions (5% CO₂-95% air) until they were approximately 75% confluent.Medium was then removed and the cells were made “serum-free” byincubation in MEM with no FBS for 24 hours. Adenosine or vehicle (MEM)was added for an additional 18 hours. This medium was then replaced withfresh MEM, and the cells were pulsed with 1 mCi/ml [³H] thymidine (6.7Ci/mmol). After a 2 hour incubation period, the medium was discarded andthe cells were rinsed twice with cold (4° C.) Hank's balanced saltsolution (HBSS) and incubated for 5 minutes with 0.5 ml cold 10% (w/v)trichloroacetic acid (TCA) . The wells were then rinsed with 8% TCA andthe TCA-insoluble material was solubilized with 0.5 ml of a solution of0.2M NaOH and 0.2% sodium decyl sulfate (SDS). The radioactivity of thisfraction was determined by standard liquid scintillation spectrometrictechniques.

Incorporation of [³H] thymidine was expressed as counts per minute (cpm)of ³H per culture. Data in each experiment was derived from 4identically treated wells. Since the cpm/well exhibited variationbetween experiments, data representing combined experiments areexpressed herein as a percent of their respective mean control value.

Incorporation of [³H]phenylalanine

Incorporation of [³H]phenylalanine was measured as an index of proteinsynthesis. Human skin fibroblasts were seeded into 24-well cultureplates in MEM containing 10% FBS. When cells had grown to approximately75% confluence the culture medium was replaced with serum-free MEM withor without adenosine. After 48 hours, 2 μCi/ml [³H]phenylalanine wasadded to the cultures. Unlabeled phenylalanine (0.36 mM) was also addedto equalize concentrations of intracellular and extracellularphenylalanine. After 8 hours, medium was removed and the cells werewashed twice with cold (4° C.) HBSS and incubated for 20 minutes in cold10% (w/v) TCA. Cells were then incubated 5 minutes in 95% ethanol (4°C.) and the TCA-insoluble material was solubilized with a solution of0.2M NaOH and 0.2% SDS. The radioactivity of this fraction wasdetermined by standard liquid scintillation spectrometric techniques.

Incorporation of [³H] phenylalanine was expressed as cpm of ³H perculture well and data in each experiment were derived from sixidentically treated wells. Since the cpm/well exhibited variationbetween experiments, data representing combined experiments areexpressed as a percent of their respective mean control value.

Determination of Cell Size

Human fibroblasts in MEM 10% FBS were seeded into 25 cm² culture flasksat a density of 1×10⁴ cells/cm². When the cells had grown toapproximately 80% confluence the culture medium was removed and thecells were incubated in serum-free MEM for 24 hours. Adenosine orvehicle (MEM) was added for 18 hours and cells were then washed twicewith cold (4° C.) HBSS. Cells were removed with 0.25% trypsin incalcium- and magnesium-free MEM and diluted in cold (4° C.) HBSS formeasurement of relative cell size with a fluorescence-activated cellsorter (FACS; Becton Dickinson Vantage). Cell size was determined byforward light scatter on a minimum of 1×10⁴ cells per experiment.

Experimental Materials

MEM, FBS, penicillin, streptomycin, trypsin, and HBSS were obtained fromGIBCO (Grand Island, N.Y.), [³H] thymidine (6.7 Ci/mmol) andphenylalanine, L-ring-2,3,4,5,6-³H] (92 Ci/mmol) were obtained fromDupont NEN (Boston, Mass.). Adenosine was from Boehringer Mannheim, SDSwas from National Diagnostics, (Highland Park, N.J.) and TCA and ethanolwere obtained from Fisher Scientific (Pittsburgh, Pa.).

Data Analysis

Analysis of variance (ANOVA) was used to determine statisticaldifferences between means. The Dunett's test was applied for multiplecomparisons as described in Zar, J. H., Biostatistical Analysis.Englewood Cliffs, N.J., Prentice Hall, Inc. pp. 150-153, 1984. Inaddition, the Wilcoxon test was employed to verify differences betweenvalues expressed as a percentage. Differences were consideredstatistically different when P<0.05.

DNA Synthesis

Exposure to 10⁻⁴M adenosine increased [³H]thymidine incorporation by43±9% in five studies on cultures of human fibroblasts (AG607720B) madequiescent by serum removal. These results are summarized in FIG. 1A. Incontrast, adenosine (10⁻⁴M) had no effect on [³H]thymidine incorporationin cultures of human lung fibroblasts (IMR-90) (FIG. 1B). Concentrationsof adenosine ranging from 10-7 M to 10⁻³M also failed to stimulate[³H]thymidine incorporation in IMR-90 lung fibroblasts (data not shown).

The effect of adenosine on DNA synthesis was additionally determined onskin fibroblast cultures from six different human donors. Adenosine(10⁻⁴M) stimulated DNA synthesis in all three cultures derived fromyoung human donors (Table 1). Values shown are means±SEM, where n isnumber of experiments. Exposure to adenosine and determination of [³H]thymidine incorporation were as described above. The asterisk denotes avalue significantly different from the corresponding control (100%).TABLE 1 Effect of adenosine on [³H] thymidine incorporation intocultured human skin fibroblasts derived from young donors [³H] thymidineincorporation Cell Adenosine Donor (% of strain (10⁻⁴ M) Age Sexcontrol) n AG07720B − 24 F 100 24 + 124 ± 7*  24 AG07306A − 28 F 100 6 +193 ± 20* 6 AG09605 − 30 M 100 12 + 133 ± 15* 12

Peak stimulation of [³H]thymidine incorporation (93±20%, n=6) wasachieved in human skin fibroblast cultures derived from a 28 year oldfemale (AG07306A).

Adenosine (10⁻⁴M) stimulated DNA synthesis in 2 of 3 cultures derivedfrom aged human donors (Table 2). As in Table 1, values are means±SEM,and n is the number of experiments performed. The asterisk denotes ameasurement significantly different from the corresponding control(100%). Adenosine exposure increased [³H]thymidine incorporation by53±31% and 54±22% in human skin fibroblast cultures derived from a 70year-old male and a 84 year-old male, respectively. Adenosine had noeffect on cultures derived from a 67-year old female. TABLE 2 Effect ofadenosine on [³H] thymidine incorporation into cultured human skinfibroblasts derived from aged donors [³H] thymidine incorporation CellAdenosine Donor (% of strain (10⁻⁴ M) Age Sex control) n AG11728 − 67 F100 6 + 91 ± 6  6 AG12949 − 70 M 100 11 + 150 ± 31* 11 AG11730 − 84 M100 10 + 154 ± 22* 10Protein Synthesis

The effect of adenosine on protein synthesis was determined by measuring[³H]phenylalanine incorporation into cultures of human fibroblasts froma young and aged donor. Cultures made quiescent by serum removal wereexposed to adenosine (10⁻⁶M to 10⁻⁴M) for 48 hours and then pulsed withphenylalanine. In skin fibroblast cultures derived from a 28-year oldfemale (AG073060A) and an 84-year old male (AG11730) adenosine(10⁻⁴M)increased protein synthesis by 13±4% (n=25) and 13±6% (n=17) ,respectively (FIG. 2).

Cell Size

The effect of adenosine on cell size was determined on human skinfibroblasts from young and aged donors by measuring forward lightscatter in a FACS analyzer. cultures made quiescent by serum removalwere exposed to adenosine for 18 hours, removed by trypsinization, anddiluted in 4° C. HBSS. A minimum of 1×10⁴ cells were measured for eachexperiment. The results are shown in Table 2. Values are mean±SEM forrelative cell size determined by forward light scatter (FLS) in afluorescence-activated cell sorter, and n=number of cells measured. Theasterisk denotes the measurement is significantly different fromcorresponding control.

In skin fibroblast cultures from a 28 year old female (AG073060A)adenosine (10⁻⁴M) significantly increased cell size by 1.8 and 2.2% intwo of three experiments (Table 3).

The effect of adenosine on cell size was also measured on skinfibroblasts from an aged donor. The results are shown in Table IV.Values are mean±SEM for relative cell size determined by forward lightscatter (FLS) in a fluorescence-activated cell sorter, where n is thenumber of cells measured. An asterisk indicates a value significantlydifferent from corresponding control.

In cultures derived from an 84-year old male (AG11730), adenosine(10⁻⁴M) significantly increased cell size by 2.7-4.9% in 3 of 3experiments (Table 4). TABLE 3 Effect of adenosine on cell size incultured human skin fibroblasts derived from young donors RelativeExperiment Adenosine Size % Number (10⁻⁴ M) (FLS) increase n 1 − 524 ±0.55 — 1.5 × 10⁴ + 526 ± 0.55 0.4  1.5 × 10⁴ 2 − 319 ± 1.24 — 1.0 ×10⁴ +  326 ± 1.16* 2.2* 1.0 × 10⁴ 3 − 342 ± 0.94 — 1.0 × 10⁴ +  348 ±0.95* 1.8* 1.0 × 10⁴

TABLE 4 Effect of adenosine on cell size in cultured human skinfibroblasts derived from aged donors Relative Experiment Adenosine Size% Number (10⁻⁴ M) (FLS) increase n 1 − 333 ± 0.79 — 1.0 × 10⁴ +  342 ±0.75* 2.7* 1.0 × 10⁴ 2 − 323 ± 1.01 — 1.0 × 10⁴ +  337 ± 0.96* 4.3* 1.0× 10⁴ 3 − 306 ± 0.81 — 1.0 × 10⁴ +  321 ± 0.81* 4.9* 1.0 × 10⁴

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention. For example, while theinvention has been described using adenosine and adenosine agonists,other compounds structurally similar to adenosine can also be used,e.g., purine-containing compounds and compounds having a ribosyl moiety.Other aspects, advantages, and modifications of the invention are withinthe scope of the following claims.

1-53. (canceled)
 54. A method for enhancing the condition of unbrokenskin of a mammal by reducing one or more of wrinkling, roughness,dryness, or laxity of the skin without increasing proliferation ofdermal cells in the skin, the method comprising topically applying tothe skin a composition comprising adenosine or an adenosine analog in anamount effective to enhance the condition of the skin without increasingdermal cell proliferation.
 55. The method of claim 54, wherein thecomposition comprises an adenosine analog in a concentration of about10⁻⁴ M to 10⁻⁷ M.
 56. The method of claim 55, wherein the adenosineanalog concentration is about 10⁻⁴ M.
 57. The method of claim 54,wherein the composition comprises adenosine in a concentration of about10⁻⁴ M to 10⁻⁷ M.
 58. The method of claim 57, wherein the adenosineconcentration is about 10⁻⁴ M.
 59. The method of claim 54, wherein thecomposition further comprises a conditioning agent.
 60. The method ofclaim 59, wherein the conditioning agent is a humectant, an emollient,or an occlusive agent.
 61. The method of claim 54, wherein the mammal isa human.
 62. The method of claim 54, wherein the skin comprises a skingraft.
 63. The method of claim 54, wherein the composition furthercomprises a transdermal delivery agent.
 64. The method of claim 54,wherein the composition is in a transdermal patch and the composition istopically applied by contacting the patch to the skin.
 65. The method ofclaim 54, wherein the adenosine analog is selected from the groupconsisting of 2′-deoxyadenosine; 2′,3′-isopropoylidene adenosine;toyocamycin; 1-methyladenosine; N-6-methyladenosine; adenosine N-oxide;6-methylmercaptopurine riboside; 6-chloropurine riboside; 5′-adenosinemonophosphate; 5′-adenosine diphosphate; 5′-adenosine triphosphate;phenylisopropyl-adenosine; 1-Methylisoguanosine; N6-cyclohexyladenosine;N6-cyclopentyladenosine; 2-chloro-N6-cyclopentyladenosine;2-chloroadenosine; adenosine amine congener; 2-p-(2-carboxy-ethyl)phenethyl-amino-5′-N-ethylcarboxamido-adenosine;N-ethylcarboxamido-adenosine; napthyl-substituted aralkoxyadenosine;5′(N-cyclopropyl)-carboxamidoadenosine; 2-chloroadenosine;N6-phenyladenosine; N6-phenylethyladenosine; and 2-phenylaminoadenosine.66. The method of claim 54, wherein the adenosine analog is 5′-adenosinemonophosphate; 5′-adenosine diphosphate; or 5′-adenosine triphosphate.67. A method for enhancing the condition of unbroken skin of a mammal byreducing one or more of wrinkling, roughness, dryness, or laxity of theskin, the method comprising topically applying to the skin a compositioncomprising an adenosine analog in an amount effective to enhance thecondition of the skin, wherein the adenosine analog concentration isabout 10⁻⁴ M to 10⁻⁷ M.
 68. The method of claim 67, wherein theadenosine analog concentration is about 10⁻⁴ M.
 69. The method of claim67, wherein the adenosine analog is selected from the group consistingof 2′-deoxyadenosine; 2′,3′-isopropoylidene adenosine; toyocamycin;1-methyladenosine; N-6-methyladenosine; adenosine N-oxide;6-methylmercaptopurine riboside; 6-chloropurine riboside; 5′-adenosinemonophosphate; 5′-adenosine diphosphate; 5′-adenosine triphosphate;phenylisopropyl-adenosine; 1-Methylisoguanosine; N6-cyclohexyladenosine;N6-cyclopentyladenosine; 2-chloro-N6-cyclopentyladenosine;2-chloroadenosine; adenosine amine congener; 2-p-(2-carboxy-ethyl)phenethyl-amino-5′-N-ethylcarboxamido-adenosine;N-ethylcarboxamido-adenosine; napthyl-substituted aralkoxyadenosine;5′(N-cyclopropyl)-carboxamidoadenosine; 2-chloroadenosine;N6-phenyladenosine; N6-phenylethyladenosine; and 2-phenylaminoadenosine.70. The method of claim 67, wherein the adenosine analog is 5′-adenosinemonophosphate; 5′-adenosine diphosphate; or 5′-adenosine triphosphate.71. A composition comprising adenosine or an adenosine analog in aconcentration effective when applied to skin of a mammal to deliver aconcentration of about 10⁻⁴ M to 10⁻⁷ M to dermal cells in the skin, andan angiogenesis factor.
 72. The composition of claim 71, wherein theangiogenesis factor is vascular endothelial growth factor or basicfibroblast growth factor.
 73. The composition of claim 71, furthercomprising a conditioning agent.